Process of isolating lactoferrin

ABSTRACT

A process for isolating lactoferrin from milk or a casein-containing component or product containing lactoferrin, comprising subjecting a precipitate of casein obtained from the milk of the casein-containing component or product, with which precipitate at least part of the milk lactoferrin is associated, to a treatment with an extraction medium whereby at least part of the lactoferrin associated with the precipitate of casein is released to the medium. When lactoferrin has been released, the medium may be subjected to different types of purification operation steps. The purification operations are selected in accordance with the purity requirements of the intended specific application. Furthermore, lactoferrin and/or a composition comprising lactoferrin according to the above-mentioned process is provided.

This application is a national phase under 35 U.S.C. § 371 of PCTInternational Application No. PCT/DK03/00135 which has an Internationalfiling date of Mar. 6, 2003, which designated the United States ofAmerica.

FIELD OF INVENTION

The present invention relates in general to the field of isolatingproteins. Specifically there is provided a novel process of isolatinglactoferrin from milk or any other casein-containing material.

TECHNICAL BACKGROUND AND PRIOR ART

Milk is one of the most thoroughly researched foods in history.Countless scientific papers document the composition of milk anddescribe the biological functionalities in this complex bio-resource.Proteins, peptides, enzymes and other biomolecular substances constitutemajor and very important component of milk and are believed to beresponsible for many of the specific functionalities passed on from amother to her new-born in addition to basic nutrients.

Casein is the colloidal protein constituent of milk which can beprecipitated e.g. by treatment with rennet enzymes under neutral orweakly acid pH conditions or by means of a mineral salt, such as acalcium salt, or by means of acids at a pH of e.g. from 4.6 to 5.6.

Casein in milk is precipitated during a number of differentdairy-related industrial processes, e.g. cheese production and casein orcaseinate production.

During cheese manufacturing, the casein is precipitated by e.g. a rennetenzyme activity and the casein coagulum (cheese curd) contains entrainedbut soluble whey proteins, fat, lactose and minerals from milk too.

Cheese whey is a relatively dilute protein solution comprising lactose,minerals and lipids that is obtained as a by-product when producing anytype of cheese by the aid of rennet enzymes and/or microbiologicalstarter cultures.

In the past two decades, there has been a significant focus onutilisation of whey proteins e.g. bovine whey proteins from milk. Today,several bovine Whey Protein Concentrates (WPC) and bovine Whey ProteinIsolates (WPI) are standard products obtained through various membranefiltration techniques as well as ion exchange adsorption procedures.

Further utilisation of the bovine whey in terms of fractionation of theproteins into individual protein fractions, such as β-lactoglobulin,α-lactalbumin, immunoglobullns, lactoperoxidase, and lactoferrin, ismade possible through chromatographic packed bed and expanded bedseparation techniques. Protein products from chromatographic separationtechnologies are generally characterised by their low- to non-fatcontent and are useful for a broad range of applications e.g. withinfoods, feeds, functional foods, and health care products.

Since the first market introductions of WPC and WPI products and morerecently, the first purified single protein products comprisinglactoferrin, an ever increasing demand for even more sophisticated andstill more efficient and cost effective production processes hasevolved.

Lactoferrin is a metallo-protein found naturally within biologicalfluids, such as milk and saliva, at mucosal surfaces and within whiteblood cells at relatively low concentrations. Besides working as atransmitter molecule in iron assimilation for the infant, lactoferrinhas an antibacterial functionality towards iron-dependentmicroorganisms.

Due to its antibacterial, anti inflammatory and immune modulatingproperties, lactoferrin offers promising new applications, for examplein the treatment of diseases, commonly difficult to treat withantibiotics.

Thus a broad range of activities and applications of products comprisinglactoferrin have been suggested:

-   -   Natural antibiotic activity—killing many disease causing        bacteria, whilst protecting the natural bacterial flora of the        body.    -   Immune-boosting activity—lactoferrin aids in maturation and        regulation of a number of immune competent cells throughout the        body.    -   Antioxidant activity—the strong binding of iron by lactoferrin,        prevents “free iron” from forming free radicals. Free radicals        have been implicated as a causative agent in many diseases        including cancers.    -   Antifungal activity—Lactoferrin kills a range of fungi and        yeast, including the causative agent of thrush, Candida        albicans.    -   Research in antiviral agents has shown that lactoferrin can        prevent viruses, such as HIV, hepatitis and CMV, from binding to        the cells of the body and thus preventing viral infection.    -   Antitumour activity—lactoferrin and related peptides have been        shown to suppress tumour growth and prevent tumour formation in        animal models.    -   Iron uptake—As lactoferrin binds very tightly to iron,        lactoferrin is believed to allow efficient uptake of iron into        the body. This is considered as being beneficial for anaemic        patients.

Furthermore, treatment of meat surfaces with lactoferrin solutions toinhibit bacterial growth and thereby minimise the risk of foodcontamination and prolong shelf life of certain food products has beensuggested as an important future application of lactoferrin.

The concentration of lactoferrin in bovine skimmed milk is usuallysmall, typically between 80-200 mg/ml depending on e.g. thepasteurisation and other pre-treatment history of the skimmed milk.After precipitation of the casein present in milk, the concentration oflactoferrin in bovine whey is typically between 10-100 mg per litredepending on the physical and chemical pre-treatment of the whey.

Thus, there is a need for an effective, simple and cost effective largescale process to enable an industrial production of lactoferrin productsto be used in various applications.

During manufacturing of certain cheese products the cheese curd iswashed with a salt solution or solid salt is added to the cheese curdwhich leads to the separation of a protein containing extract. Theaddition of salt or saturated salt solution to the cheese curd resultsin syneresis of the curd causing expulsion of water, whey components(fat, protein, lactose, etc) and some salt.

Regarding the nature of such an extract, U.S. Pat. No. 5,783,237discloses such an extract that is discharged even though it containsvaluable milk and whey components since it cannot be usefullyreprocessed because of the high salt content and it is difficult todispose of because of its high Biological Oxygen Demand (BOD) andsalinity level.

Thus, the extract is at many production sites regarded as a wasteproduct that has little or no value and in most instances it is aproblem to discard the product. Furthermore, it seems to be a generalobservation that the extract cannot be further processed to takeadvantage of any valuable compounds present therein due to the high saltconcentration.

Casein and caseinates are generally produced from skimmed milk. It isprecipitated by treating milk with a destabilising agent, such as anacid, a mineral salt or rennet and then separated from the residual wheyby means of a separator or filter. It is then typically washed withlarge quantities of water and dried.

U.S. Pat. No. 4,976,865 discloses a method of separating biologicalmacromolecules by using packed bed chromatography as well as expandedbed chromatography which is economically feasible to provide anindustrial scale production. The compounds are primarily obtained fromnatural products such as milk. In particular, the method disclosed maybe applied to the chromatographic separation of whey proteins,specifically are β-lactoglobulin and α-lactalbumin are extracted from a“mild” whey i.e. whey without increased salt concentration or ionicstrength. For elution of β-lactoglobulin and α-lactalbumin a 0.1Nhydrochloric acid solution is used. The method does not discloseisolation and purification of lactoferrin. Furthermore, the whey usedfor isolation and purification of whey proteins is a mild whey and theuse of an extract obtained from the treatment of precipitated caseinwith a salt or a salt solution has not been disclosed.

U.S. Pat. No. 4,791,193 discloses a process for producing lactoferrrinwith a high purity from a bovine source. It is disclosed that in spiteof the useful physiological functions of lactoferrin, it has beendifficult to isolate and purify lactoferrin from milk at an industrialscale because of its minor proportion in milk. The milk used in thisinvention is in the form of either skimmed milk or as whey. The patentdiscloses a process utilising a solution of one or more salts selectedfrom the group consisting of sodium chloride, potasium chloride, calciumchloride and magnesium chloride for eluting the whey protein,lactoferrin, captured by a weakly acidic cation-exchanger. Theconcentrations of the salt solutions applied for elution are in therange of 0.4-2.5 and 1.5-12. U.S. Pat. No. 4,791,193 does not discloseuse of an extract for isolation of whey proteins such as lactoferrin.Additionally, the process does not disclose the preparation of anextract obtained from the precipitated milk casein for the refinement oflactoferrin.

U.S. Pat. No. 5,596,082 discloses a process for isolating lactoferrinand lactoperoxidase from milk and milk products and products obtained bysuch a process. In particular, a process is disclosed for isolatinglactoferrin and lactoperoxidase from milk at an industrial scale. Thestarting material used is whey from cheese manufacturing or neutralisedcasein whey at pH 6.5 obtained from skimmed milk after caseinprecipitation. Lactoferrin and lactoperoxidase are adsorbed to a cationexchanger at a high surface velocity and subsequently eluted from thecation exchanger with a salt solution. This patent does not disclose theuse of an extract for refining whey proteins such as lactoferrin.Additionally, the process does not disclose the preparation of anextract obtained from the precipitated milk casein for the isolation oflactoferrin.

The above-mentioned processes are based on known chromatographictechniques wherein a lactoferrin-containing liquid passes a solid phasedesigned to capture lactoferrin by a selective binding.

A disadvantage of the known production processes for lactoferrin is therelatively low concentration of the lactoferrin naturally present inmilk and whey raw materials. This implies that large volumes of liquidmust be treated in order to produce a certain amount of lactoferrin. Therelatively high concentration of other milk/whey proteins makes itchallenging and relatively expensive to obtain a product with a highdegree of lactoferrin purity.

Furthermore, the preparation of an extract obtained from theprecipitated milk casein for the isolation of lactoferrin has not beendisclosed in the prior art.

The inventors of the present invention surprisingly found that treatmentof the precipitated casein, from e.g. cheese manufacturing, may resultin an extract containing an elevated concentration of lactoferrinrelative to traditional whey obtained as a waste product in e.g. cheesemanufacturing. This lactoferrin-enriched extract is suitable for anefficient recovery of lactoferrin or alternatively, directly applicablein low cost applications on wet or dry basis.

Thus, by providing a process according to the present invention it hasbecome possible to recover whey proteins, such as lactoferrin, in amanner which is cheap, specific, more efficient, easily performed andthe invention is easily compatible with automated and semi-automatedsystems.

SUMMARY OF THE INVENTION

Accordingly, it is a primary objective of the present invention toprovide a process for isolating lactoferrin from precipitated caseinobtained from milk or any other casein-containing component or product.During said casein precipitation, proteins, fat, lactose and othersoluble components are associated to the precipitated casein structure.One of the soluble proteins entrapped in the precipitated casein islactoferrin. This protein may be extracted by using a specificallydesigned extraction medium comprising a salt, acid or base or acombination hereof to release the soluble lactoferrin to the medium.

When lactoferrin has been released, the medium may be subjected tofurther steps of purification operations depending on the nature ofwhich depends on the planned application of the isolated lactoferrin.The purification operation steps are selected in accordance with thepurity requirements of the specific intended application.

It is an object of the present invention to provide a simple and lowcost process for the industrial production of lactoferrin from milk or acasein-containing component or product containing lactoferrin. Thus, itis an object of the present invention to provide lactoferrin products atrelatively low costs to enable commercial uses of such products thathave hitherto been inhibited by high production costs and also toprovide added value products derived from the extract and thereby allowfor more efficient use of the natural resources.

Additionally, it is an object of the present invention to provide a costeffective process for production of lactoferrin products from an extractobtained from precipitated casein and other liquid fractions that may beobtained in the context of any type of industrial casein precipitation.

Accordingly, in the broadest aspect of the present invention a processis provided for isolating lactoferrin from milk or a casein-containingcomponent or product containing lactoferrin, comprising subjecting aprecipitate of casein obtained from the milk or the casein-containingcomponent or product, with which precipitate at least part of the milklactoferrin is associated, to a treatment with an extraction mediumwhereby at least part of the lactoferrin associated with the precipitateof casein is released to the medium.

It is a further aspect of the present invention to provide lactoferrinand/or a composition comprising lactoferrin according to theabove-mentioned process.

DETAILED DISCLOSURE OF THE INVENTION

In the present context the term “casein” relates to a protein mainlyfound in milk, the casein may be naturally or synthetically produced andmay be precipitated in a form where it is associate with lactoferrin.

At least under certain pH and ionic strength conditions a part of thelactoferrin present in the milk is loosely associated with casein, whenit is precipitated by a treatment of the milk e.g. during production ofcheese or caseinates. It is assumed that this association of lactoferrinis preferential in relation to the other soluble milk proteins that stayin solution during casein precipitation and are subsequently found inthe liquid whey fraction.

In the present context, the term “precipitation” or “precipitate”relates to any kind of joining between casein particles. The joining maybe by actions such as electrostatic action, ionic interaction, covalentinteraction and formation of hydrogen bonds. The joining between theparticles may provide a solid.

The process of precipitating casein may be initiated by changes in pHand/or temperature of the milk or a casein-containing component orproduct and/or by adding a coagulating agent hereto. The coagulatingagent may be selected from the group of agents consisting of rennet,microorganisms, salts, acids and bases.

In the present context, the term “milk or a casein-containing componentor product” relates to milk in the form of skimmed milk, dried milkpowder, fully natural milk or any type of low fat milk. It may also be amicrofiltrated milk wherein the casein is enriched (the retentate)relative to the whey proteins that are passing through the microfilter(the permeate) but also concentrated milk e.g. skimmed milk concentratedby membrane filtration or any kind of composition comprising casein. Themilk or the casein-containing component or product may be either a solidor a liquid.

The milk or the casein-containing component or product are suitable rawmaterials in the production of lactoferrin according to the inventionand the milk or a casein-containing component or product may becollected from an animal or a transgenic animal capable of producinghuman lactoferrin in their milk. The animal or the transgenic animal maye.g. be selected from the group consisting of herd animals, cows,camels, buffaloes, pigs, horses, deer, sheep, goats, dogs and cats. Inthe case where the product may be produced by a transgenic animal,according to the invention the milk or the casein-containing componentor product will comprise human lactoferrin.

In the present context, the term “associated” relates to the manner inwhich latcoferrin is connected or bound to the casein precipitate sothat lactoferrin is retained from leaving the precipitated casein. Thisconnection may be provided by adsorbtion or fixation in any other way oflactoferrin to the precipitated casein, or lactoferrin may be entrappedor captured within the precipitated casein structure.

In the present invention “artificial milk” wherein soluble casein hasbeen added to e.g. a liquid whey fraction obtained from an independentsource can be used. In this case the liquid whey fraction may be aconcentrated and optionally a desalted whey fraction.

Assumed herein the term “artificial milk” relates to separate caseincomponents or casein products naturally or synthetically produced whichmay be precipitated and used to associate with lactoferrin.

In one embodiment of the present invention, the casein is added to aliquid whey fraction as an insoluble form of casein still able toassociate with lactoferrin. The casein may be separated from the whey,regenerated and recycled several times in the same way as an ionexchanger might be used in many processes.

In principle, any type of casein precipitate leading to an associationof lactoferrin according to the invention may be applied. Methods ofcasein precipitation is typically based on treating the milk with adestabillsing agent, such as an acid, a mineral salt or a rennet enzymee.g. chymosin and/or pepsin or other proteolytic enzymes. Furthermore,microorganisms producing various proteolytic enzymes may be used forprecipitation of the milk caseins.

When acid precipitation is carried out a low cost mineral acid such ashydrochloric acid, phosphoric acid and sulfuric acid is preferred.However, food grade organic acids such as acetic, citric and lactic acidmay be particularly preferred. The pH of the milk is preferably adjustedto or below the point of casein precipitation which would typically takeplace in the range of pH 6 to pH 4 depending on the presence of anyadditives in the milk.

In a preferred embodiment of the present invention, the optimaladsorption or other type of fixation of lactoferrin to the caseinprecipitate takes place in the pH range of 4-7, e.g. in the range of4.5-6.5, such as in the range of 5-6.

The highest optimal association of lactoferrin to the casein precipitatetakes place at the lowest possible ionic strength.

In one preferred embodiment of the present invention the ionic strengthis kept at or close to the ionic strength of the native milk. However,still more preferred is an ionic strength in the range of 1-75% of thenative milk and most preferred is an ionic strength in the range of1-25% of the native milk for the association of lactoferrin to theprecipitated casein or any other kind of ion exchange system.

When the casein or parts of the casein present in the milk or acasein-containing component or product are precipitated the remainingliquid, whey, comprises soluble proteins, fat, lactose and other wheycomponents. This precipitated casein is separated from the whey and iscontacted with a washing solution to wash out at least part of undesiredentrained whey proteins, fat and lactose from the precipitated milkcasein and the washing solution is subsequently drained off theprecipitated milk casein.

This washing procedure of the casein precipitate is an optional step andin a preferred embodiment of the present invention, the optional washingprocedure is based on an aqueous washing solution that does notinterfere with the association of lactoferrin with the caseinprecipitate. The washing solution is typically selected from the groupconsisting of tap water, demineralised water, filtered tap water,diluted salt solutions, diluted buffer solutions or combinations hereof.Lactoferrin associates with the precipitated casein is subsequentlyreleased from the casein precipitate by contacting the precipitatedcasein with an extraction medium. The resulting separated lactoferrinintermediate will be of a significantly higher purity than without thewashing step and the separated lactoferrin intermediate may require aless intensive refinement procedure in order to obtain a properlactoferrin product.

In a preferred embodiment of the present invention, the washing solutioncomprises a dilute salt solution selected from the group consisting ofsodium chloride or any other low cost, food grade mineral or organicsalt at a concentration less than a concentration capable of releasingthe lactoferrin from the casein precipitate. The concentration of thedilute salt solution is typically at the most 0.1 N, at the most 0.05 Nor at the most 0.02 N.

In another preferred embodiment of the present invention the washingsolution comprises a dilute buffer solution which comprises acetic,citric, lactic or any other organic acid buffer of low cost and foodgrade quality at a concentration of at the most 0.1 N, at the most 0.05N or at the most 0.02 N.

In a preferred embodiment of the present invention, the washing solutioncan be tailor-made in order to obtain a high-grade stream oflactoferrin. Initially, the precipitate is washed with a buffer torelease other milk-derived molecules. Subsequently, the precipitate iswashed with a buffer to release lactoferrin in a purer state. Thisbuffer can be a salt containing buffer or a buffer with a specific pHvalue or a buffer containing a specific composition to release thelactoferrin selectively. In this way, a purer lactoferrin is obtainedand the number of unit operations necessary to isolate lactoferrin maybe limited.

Subsequently, it was surprisingly found that precipitated casein is ableto associate with lactoferrin and precipitated casein becomes availableto the production of lactoferrin products by releasing lactoferrin fromthe casein precipitate by the aid of an extraction medium. In thepresent context, the term “extraction medium” relates to the medium thatleads to the release of lactoferrin adsorbed or fixed to a caseinprecipitate.

The extraction medium may be any type of liquid or solid capable ofreleasing associated lactoferrin from a casein precipitate.

In a preferred embodiment of the present invention, the extractionmedium provides a change In one or more of the parameters selected fromthe group consisting of pH, ionic strength, salt content,hydrophobicity, charge, temperature and pressure. This change may beobtained by using an extraction medium selected from the groupconsisting of inorganic and organic salts, acids or bases. In apreferred embodiment of the present invention, the extraction mediacontains inorganic salts of food grade such as sodium chloride,potassium chloride, calcium chloride, sodium sulphate; organic saltssuch as sodium acetate, sodium citrate, sodium lactate and acids such ashydrochloric acid, phosphoric acid, sulphuric acid, acetic acid, citricacid and/or lactic acid.

The properties of the extraction medium may be influenced by changingone or more of the above mentioned parameters. These changes mayinfluence the conductivity of the extraction medium. In the presentcontext the term “conductivity” relates to the ability of the extractionmedium to conduct an electrical current. In an embodiment of the presentinvention, the extraction medium after extraction of precipitated caseinhas a conductivity of at least 5 mS/cm, such as at least 10 mS/cm, e.g.at least 20 mS/cm, such as at least 30 mS/cm, e.g. at least 50 mS/cm,such as at least 75 mS/cm, e.g. at least 100 mS/cm, such as at least 125mS/cm, e.g. at least 150 mS/cm, such as at least 200 mS/cm, e.g. atleast 300 mS/cm.

In a preferred embodiment of the present invention, the extractionmedium employed is a salt and the salt is added to the caseinprecipitate either as a solid or as a salt solution. When the salt isadded as a solid the resulting syneresis will lead to the separation ofa liquid wherein the released lactoferrin may be found. The addition ofsolid salt would result in a final salt concentration in the separatedliquid in the range of 0.01-5N, such as 0.05-5N, e.g. 0.1-4N, such as0.1-3N, e.g. 0.1-2N, such as 0.1-1.5N, e.g. 0.1-1N, such as 0.2-3N, e.g.0.3-3N, such as 0.2-1.5N, e.g. 0.2-1.2N such as 0.3-1.5N, e.g. 0.3-1N.When the salt is added as a salt solution, the salt solution has aconcentration in the range of 0.01-5N, such as 0.05-5N, e.g. 0.1-4N,such as 0.1-3N, e.g. 0.1-2N, such as 0.1-1.5N, e.g 0.1-1N, such as0.2-3N, e.g. 0.3-3N, such as 0.2-1.5N, e.g. 0.2-1.2N such as 0.3-1.5e.g.0.3-1N.

In another preferred embodiment of the present invention the extractionmedium comprises an acid and the resulting pH of the liquid extractedand separated from the casein precipitate after contacting with the acidis in the range of pH 1-5, such as pH 1-4.5, e.g. pH 2-4, such as pH2-3, e.g. pH 3-4.

Alternatively, the extraction medium may be applied directly to theprecipitated casein without prior washing of the casein precipitate. Inthis case, the extraction medium may contains the soluble whey proteins,lipids and lactose which have also been entrained in the caseinprecipitate together with the released lactoferrin. In this instance, alactoferrin intermediate will be more impure and may require a moreintensive refinement procedure in order to obtain a proper lactoferrinproduct. However, it has been found that even so the lactoferrinintermediate will be significantly enriched in respect of lactoferrinrelative to the lactoferrin present in the ordinary liquid whey fractionthat is used for lactoferrin production in the processes disclosed inthe prior art.

The extraction medium comprising the released lactoferrin is separatedfrom the casein precipitate as a lactoferrin intermediate and thelactoferrin may be isolated from the extraction medium to obtain alactoferrin product.

Separation and collection of the liquid lactoferrin intermediate fromthe casein precipitate will be performed by using methods known per see.g. draining, centrifugation, filtering, filter pressing.

In a preferred embodiment of the present invention, the subjecting ofthe precipitated casein to fresh extraction medium may be repeated oneor several times to ensure an optimal recovery of lactoferrin entrappedwithin the casein precipitate.

The isolation methods chosen to obtain a lactoferrin product will dependon the quality specifications for a particular use or of the particularlactoferrin product. Thus, applications within health care andpharmaceutical applications are likely to require very high purity ofthe lactoferrin product while other applications such as the treatmentof meat surfaces to inhibit bacterial growth may have significantlylower demand for high purity lactoferrin products.

The isolation methods used in the present invention may thus be chosenamong the methods known per se for isolating a crude productintermediate to a final, stabilised product with known standardisedquality characteristics. These methods may be used alone or incombination and include unit operations such as:

-   -   clarification comprising e.g. filtration, membrane filtration,        centrifugation, sedimentation or a any combination hereof,    -   concentration comprising e.g. membrane filtration including        ultrafiltration, nanofiltration, reversed osmosis, evaporation        and precipitation methods or any combination hereof, and    -   purification comprising e.g. adsorption techniques including ion        exchange, hydrophobic adsorption, affinity adsorption, mixed        mode ligand adsorption and metal chelate adsorption, gel        filtration and/or precipitation methods or any combination        hereof.

The purification operation may be performed in either an expanded bedmode and/or in a packed bed mode.

In a preferred embodiment of the present invention, the linear flow rateof the extraction medium when loaded on to the chromatography column isat least 3 cm/min, such as at least 5 cm/min, e.g. at least 8 cm/min,such as at least 10 cm/min, e.g. at least 15 cm/min, such as at least 20cm/min, e.g. at least 25 cm/min, such as at least 50 cm/min, e.g. atleast 100 cm/min.

In yet another embodiment of the present invention, the adsorbent bindslactoferrin from the extract medium after extraction of the precipitatedcasein (ml extract per ml adsorbent) in a ratio of at least 1:5, such asat least 1:10, e.g. at least 1:20, such as at least 1:30, e.g. at least1:40, such as at least 1:50, e.g. at least 1:60, such as at least 1:75,e.g. at least 1:100, such as at least 1:150, e.g. at least 1:200, suchas at least 1:300.

In a preferred embodiment of the present invention the isolation oflactoferrin may be performed by either a batch operation or by acontinuous operation.

In order to isolate lactoferrin from the extraction medium the resinused in the adsorption technique is generally equilibrated to favouradsorption or fixation of lactoferrin to the resin. In a preferredembodiment of the present invention the resin present in the adsorptiontechnique is equilibrated to a pH range of pH 3-7, such as pH 3.5-6.7,e.g. pH 4-6.6, such as pH 4.5-6.5, e.g. pH 5-6.4, such as pH 5-6.

Elution of the adsorbed lactoferrin from the resin used in theadsorption technique is obtained by increasing the pH. In a preferredembodiment of the present invention the pH for elution of lactoferrin isin the range of pH 8-13, such as pH 8.5-12, e.g. pH 8.5-11, such as pH9-10, e.g. pH 9.5-10.

In the present context, the term “elution” relates to the liquid used torelease lactoferrin adsorbed or fixed to a resin used in an adsorptiontechnique.

In a preferred embodiment of the present invention, the amount oflactoferrin isolated relative to the amount of lactoferrin present inthe milk or a casein-containing component or product before caseinprecipitation, is at least 5%, at least 10%, at least 20% at least 30%,at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90% at least 92%, at least 94%, at least 95%, at least 97% or atleast 100%.

In another preferred embodiment of the present invention, the isolationof lactoferrin present in the precipitated casein is at least 5%, atleast 10%, at least 20% at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 80%, at least 90% at least 92%, atleast 94%, at least 95%, at least 97% or at least 100%.

Alternative procedures for isolation of lactoferrin from an extractionmedium as provided by the present invention may easily be developed e.g.the procedure disclosed in the co-pending Danish patent application No.PA 2001 00869 may be applied for isolation of lactoferrin and thisapplication is hereby incorporated by reference.

In a preferred embodiment of the present invention, lactoferrin isisolated from a casein precipitate obtained from the dairy industry.

In another preferred embodiment of the present invention, lactoferrin isisolated from a casein precipitate, said casein precipitate is obtainedfrom a casein and/or caseinate production and/or from a cheeseproduction.

Stabilisation methods are provided for optimising the storage stabilityof the final lactoferrin product in terms of bacterial growth,minimising the deterioration due to protease activities and generalchemical instability of the product.

In a further preferred embodiment of the present invention, astandardisation of the lactoferrin product is performed in terms of e.g.component concentration, lactoferrin activity and colour of the product.

Lactoferrin prepared according to the process of the present inventionmay be applied in a wide array of known applications ranging from lowcost food additive to high value pharmaceutical applications.

The extract medium may after extraction of lactoferrin from theprecipitated casein, be used directly as a lactoferrin product, or theextract medium may be subjected to sterilisation and/or a minimum ofadditional preparation steps and subsequently be used, for preservationof food products preferably meat products.

The present invention will be further illustrated in the followingfigures and non-limiting examples.

FIG. 1 shows the protein composition analysed by SDS-PAGE oflactoferrin. S1 shows the supernatant removed by decanting afterprecipitation of the casein, S2 shows the supernatant obtained aftercentrifugation of the precipitated casein, S3 and S4 shows thesupernatant of two washing operations and S5 shows the extraction buffercomprising 1N NaCl removed by centrifugation.

FIG. 2 shows the protein composition of the extraction medium afterextraction of the precipitated casein analysed by SDS-PAGE. Lane 1 showsa molecular weight marker and lane 2 shows the extraction medium afterextraction of the precipitated casein.

FIG. 3 shows the isolation of lactoferrin at pH 4.0 and 4.5. Lane 1shows the extraction medium after extraction of the precipitated casein,lane 2 shows a flow through fraction at pH 4.0, lane 3 shows thesupernatant from a wash with water, lane 4 shows the supernatant from awash with SDS buffer, lane 5 shows the eluted lactoferrin, lane 6 showsa flow through fraction at pH 4.5, lane 7 shows the supernatant from awash with water, lane 8 shows the supernatant from a wash with SDSbuffer, lane 9 shows the eluted lactoferrin.

FIG. 4 shows the isolation of lactoferrin at pH 4.9. Lane 1 shows amolecular weight marker, lane 2 shows the extraction medium afterextraction of the precipitated casein, lane 3 shows a flow throughfraction at pH 4.9, lane 4 shows the supernatant from a wash with water,lane 5 shows the supernatant from a wash with SDS buffer, lane 6 showsthe eluted lactoferrin.

FIG. 5 shows the isolation of lactoferrin using expanded bed adsorptionusing a flow rate of 10 cm/min. Lane 1 shows the extraction medium afterextraction of the precipitated casein, lane 2 shows a flow throughfraction after 300 ml loaded, lane 3 shows a flow through fraction after600 ml loaded, lane 4 shows a flow through fraction after 900 ml loaded,lane 5 shows a flow through fraction after 1180 ml loaded, lane 6 showsa pool of the flow through fractions, lane 7 shows the supernatant froma wash, lane 8 shows the eluted lactoferrin.

FIG. 6 shows the isolation of lactoferrin using expanded bed adsorptionwherein the flow rate has been increased to 15 cm/min. Lane 1 shows theextraction medium after extraction of the precipitated casein, lane 2shows a flow through fraction after 300 ml loaded, lane 3 shows a flowthrough fraction after 600 ml loaded, lane 4 shows a flow throughfraction after 900 ml loaded, lane 5 shows a flow through fraction after1180 ml loaded, lane 6 shows a pool of the flow through fractions, lane7 shows the supernatant from a wash, lane 8 shows the elutedlactoferrin.

FIG. 7 shows the isolation of lactoferrin using expanded bed adsorptionusing an increased load ratio. Lane 1 shows the extraction medium afterextraction of the precipitated casein, lane 2 shows a flow throughfraction, lane 3 shows the supernatant from a wash, lane 4 shows theeluted lactoferrin.

FIG. 8 shows the isolation of lactoferrin using expanded bed adsorptionusing different conductivity values and un-diluted extracts. Lane 1shows a flow through fraction after a loading ratio of 1:15, lane 2shows a flow through fraction after a loading ratio of 1:30, lane 3shows a flow through fraction after a loading ratio of 1:45, lane 4shows the raw material.

FIG. 9 shows the isolation of lactoferrin using expanded bed adsorptionusing different conductivity values and extracts diluted 2 times. Lane 1shows a flow through fraction after a loading ratio of 1:12.5, lane 2shows a flow through fraction after a loading ratio of 1:25, lane 3shows a flow through fraction after a loading ratio of 1:37.5, lane 4shows a flow through fraction after a loading ratio of 1:50, lane 5shows the raw material.

FIG. 10 shows the isolation of lactoferrin using expanded bed adsorptionusing different conductivity values and extracts diluted 3 times. Lane 1shows the extract medium, lane 2 shows a flow through fraction after aloading ratio of 1:15, lane 3 shows a flow through fraction after aloading ratio of 1:30, lane 4 shows a flow through fraction after aloading ratio of 1:45.

FIG. 11 shows the isolation of lactoferrin using expanded bed adsorptionusing different conductivity values and extracts diluted 3 times. Lane 1shows the extract medium, lane 2 shows a flow through fraction after aloading ratio of 1:12.5, lane 3 shows a flow through fraction after aloading ratio of 1:25, lane 4 shows a flow through fraction after aloading ratio of 1:37.5, line 5 shows a flow through fraction after aloading ratio of 1:50.

FIG. 12 shows the isolation of lactoferrin using expanded bed adsorptionusing a reduced temperature. Lane 1 shows the extraction medium afterextraction of the precipitated casein, lane 2 shows a flow throughfraction after 300 ml loaded, lane 3 shows a flow through fraction after600 ml loaded, lane 4 shows a flow through fraction after 900 ml loaded,lane 5 shows a flow through fraction after 1180 ml loaded, lane 6 showsthe supernatant from a wash, lane 7 shows the eluted lactoferrin.

EXAMPLES Example 1 Isolation of Lactoferrin from Precipitated Skim Milk.

Skim milk pasteurised at 72° C. was adjusted to pH 4.5 with 1N HCl underthorough stirring and left overnight at 4-8° C. to let the precipitatedcaseins sediment.

The next day most of the supernatant (S1) was removed by decanting and asample of the loose precipitate (10 ml) was withdrawn of the caseinprecipitate and centrifuged for 5 minutes at 2000 G.

The supernatant (S2) from the centrifuged sample was removed and thecasein pellet (approx 2 ml) was washed by adding 8 ml 10 mN sodiumacetate buffer pH 4.5, mixed carefully with the precipitated caseinfollowed by repeated centrifugation for 5 minutes at 2000 G. The washwith sodium acetate buffer was repeated twice and the supernatants fromthe washing steps were labelled S3 and S4.

After the second wash the casein pellet was added 1 ml 3N NaCl to get afinal concentration of 1N NaCl and following thorough mixing the samplewas centrifuged for 5 minuets at 2000 G and the supernatant (S5) wasremoved.

All samples S1-S5 were analysed by SDS polyacrylamide electrophoresis(SDS PAGE) to detect and estimate the amount and purity of lactoferrinpresent in each sample.

SDS Page

For SDS PAGE, invitrogen SDS Page 4-20% Tris-Glycine gel (cat no.EC6025) was used. Sample preparation: 25 μl sample and 25 μl samplebuffer Tris-Glycine Invitrogen (cat no. LC2676) was mixed and boiled for5 minuets in a water bath. The running buffer 0.024M Tris (Sigma T1378),0.19M Glycine (Merck 5001901000), 0.1% SDS (Sodium dodecyl sulphate, JTBaker 2811) pH 8.6 was added.

20 μl sample was applied in each analysis slot and the power wasadjusted to give a current of 40 mA. When the blue line from the samplebuffer reached one cm from the bottom of the gel the power was turnedoff and the gel was stained overnight in Invitrogens Colloidal BlueStaining Kit (cat. no. LC 6025) on a shaking table. The next day the gelwas transferred into water and de-stained in water for 2 hours.

Results

From the SDS PAGE, FIG. 1, it is clearly seen that lactoferrin isselectively retained in the casein precipitate when washing with 10 mNsodium acetate pH 4.5 and is released (eluted) into the 1N sodiumchloride solution after washing. The eluted lactoferrin (S5) has asignificantly higher purity relative to other milk/whey proteins thanthe purity in the liquid whey fraction (S1) obtained after precipitationof the caseins.

Example 2 Isolation of Lactoferrin from the Extract Medium AfterExtraction of the Precipitated Casein, by Using Packed BedChromatography.

Refining of the extract medium after extraction of the precipitatedcasein comprising lactoferrin (LF) with packed bed adsorbentchromatography:

The extract medium after extraction of the precipitated casein wasobtained from a local cheese production and where analysed to have a pHof 5.5 and a conductivity of 54.5 mS/cm.

As can be seen on the analysis result the extract medium afterextraction of the precipitated casein has a very high concentration oflactoferrin (compare e.g. to whey as obtained from skim milk in example1, FIG. 1. Lane 1).

Experiments were performed to isolate lactoferrin by adsorptionchromatography from the extract medium after extraction of theprecipitated casein at three different pH-values.

Adsorbent

XpressLine Pro A, UpFront Chromatography A/S,

The adsorbent comprises an aromatic acid ligand and generally bindsproteins in the pH-range of pH 4 to 6 and the bound proteins arereleased by increasing the pH to 9-10 in an elution buffer.

Pre-Treatment of the Extract Medium After Extraction of the PrecipitatedCasein

The extract medium after extraction of the precipitated casein waspassed through Celite filter help (fine grade) to remove particulatematter. The pH in salt the extract was adjusted to respectively pH 4.9,pH 4.5 and pH 4.0 with 1N hydrochloric acid.

The experiments were performed in packed bed columns (Ø=0.5 cm).

The columns were packed with 1 ml of adsorbent and equilibrated withdemineralised water.

The extract medium after extraction of the precipitated casein (havingdifferent pH values) was loaded onto three individual columns with alinear flow rate of 5 cm/min.

The respective columns were washed with 10 ml demineralised water.

Bound impurities were eluted with 10 ml 10 mN sodium citrate, 0.04 mg/mlsodium dodecylsulphate (SDS) pH 5.75.

The lactoferrin was eluted from the resin with 10 ml 20 mN sodiumhydroxide.

The content of protein in each fraction was determined with SDS-PAGE(non-reduced, 4-20% tris-glycine gradient gel, Coomassie stained). SeeFIGS. 3 and 43 below.

Results

The protein concentration in the eluates has been measuredspectrophotometrically at 280 nm:

OD 280 nm Load pH in eluate mg LF in eluate Purity 4.0 2.66 12 ++(approx. 60%) 4.5 2.36 16 +++ (approx. 75%) 4.9 1.84 15 ++++ (approx.90%)

Calculation of amount lactoferrin in eluate:(OD 280 in eluate×volume of eluate×purity)/OD _(0.1%, 280 nm) OD_(0.1%, 280 nm)=1.1

The adsorbent binds lactoferrin from the extract medium after extractionof the precipitated casein in a ratio of at least 1:20 (20 ml extractper ml adsorbent). The capacity of the adsorbent towards LF increaseswhen pH increases from pH 4.0 to 4.9.

The purity of the product (lactoferrin) increases when the binding pHgoes from 4.0 to 4.9.

The resin binds bovine serum albumin (BSA) at all three pH values. It ispossible to wash out BSA with 10 mN sodium citrate, 0.04 mg/ml sodiumdodecylsulphate (SDS) pH 5.75.

The resin binds a minor fraction of β-lactoglobulin (β-LG) at pH 4.0 and4.5 but not at 4.9.

Example 3 Isolation of Lactoferrin from the Extract Medium AfterExtraction of the Precipitated Casein, by using Expanded Bed Adsorption.

Refining of the extract medium after extraction of the precipitatedcasein comprising lactoferrin (LF) with expanded bed adsorptionchromatography at 25° C.:

An extract medium after extraction of precipitated casein was obtainedfrom a cheese production facility and was analysed to have a pH of 5.6and a conductivity of 53 mS/cm. The extract medium was obtained bywashing the initially obtained cheese curd (precipitated casein) with asolution of sodium chloride whereby a solution of lactoferrin enrichedsalty whey was obtained by draining.

Adsorbent

FastLine SALTO, product number 1233-0100 UpFront Chromatography.

The adsorbent is agarose based with tungsten carbide particlesincorporated resulting in a density of approximately 2.8 g/ml. Theparticle size is in the range of 40-200 μm.

The adsorbent comprises an aromatic acid ligand and generally bindsproteins in the pH range of pH 4-6 and the bound proteins are releasedby increasing the pH to 9-10 in an elution buffer.

Pre-Treatment of the Extract Medium After Extraction of the PrecipitatedCasein

The pH in the salt extract was adjusted to 4.5 with 1N hydrochloric acidand heated to 25° C.

The experiment was performed in a FastLine®10 expanded bed column (Ø=1cm) product number 7010-0000, UpFront Chromatography.

The column was packed with 25 cm of adsorbent (19.6 ml) and equilibratedwith deminerallsed water, 25° C.

The extract medium after extraction of the precipitated casein (saltywhey) pH 4.5 was loaded onto the column with a linear flow rate of 10cm/min. 1180 ml salty whey was loaded. The flow through was collected infractions of 300 ml.

Bound impurities were eluted with 50 mN boric acid pH 9.0 (210 ml).

The lactoferrin was eluted from the adsorbent with 113 ml of 20 mNsodium hydroxide.

The content of protein in each fraction was determined with SDS-PAGE(non-reduced 4-20% tris-glycine gradient gel, Coomassie stained) SeeFIG. 5.

Results

The protein concentration in the eluate was measuredspectrophotometrically at 280 nm:

OD 280 nm in mg LF Adsorbent capacity eluate in eluate mg LF/mladsorbent Purity 4.69 477 24.3 ++++ (approx. 90%)

Calculation of mg LF in eluate:(OD 280 in eluate×volume of eluate×purity)/OD _(0.1%, 280 nm) OD_(0.1%, 280 nm)=1.1

The adsorbent binds the lactoferrin from the extract medium afterextraction of the precipitated casein in a ratio of at least 1:60 (60 mlextract per ml adsorbent). No LF is detected in the flow throughfractions. See FIG. 5.

Example 4 Isolation of Lactoferrin from the Extract Medium AfterExtraction of the Precipitated Casein, by Using Expanded Bed Adsorptionand Increased Loading Flow-Rate.

Refining of the extract medium after extraction of the precipitatedcasein comprising lactoferrin (LF) with expanded bed adsorptionchromatography. The experiment is performed at a linear flow rate of 15cm/min.

The extract medium after extraction of the precipitated casein wasobtained from a local cheese production and was the same as in example3.

All conditions except for the flow rate were the same as described inexample 3.

Results

The lactoferrin was eluted from the adsorbent with 190 ml of 20 mNsodium hydroxide.

The content of protein in each fraction was determined with SDS-PAGE(non-reduced 4-20% tris-glycine gradient gel, Coomassie stained) SeeFIG. 6 below.

The protein concentration in the eluate was measuredspectrophotometrically at 280 mM:

OD 280 nm in mg LF Adsorbent capacity eluate in eluate mg LF/mladsorbent Purity 2.68 458 23.4 ++++ (approx. 90%)

No LF was detected in the flow through fractions. See FIG. 6.

Example 5 Isolation of Lactoferrin from the Extract Medium AfterExtraction of the Precipitated Casein, by Using Expanded Bed Adsorptionand Increasing Load Ratio.

Refining of the extract medium after extraction of the precipitatedcasein comprising lactoferrin (LF) with expanded bed adsorptionchromatography. The experiment is performed at a load ratio of 1:100(100 ml of raw material per ml of adsorbent) and a linear flow rate of15 cm/min.

The extract medium after extraction of the precipitated casein wasobtained from a local cheese production and were the same as in example3.

The extract medium after extraction of the precipitated casein pH 4.5was loaded onto the column with a linear flow rate of 15 cm/min. 1960 mlwas loaded. All other conditions were as described in example 3.

Results

The lactoferrin was eluted from the adsorbent with 176 ml of 20 mNsodium hydroxide.

The content of protein in each fraction was determined with SDS-PAGE(non-reduced 4-20% tris-glycine gradient gel, Coomassie stained) SeeFIG. 7 below.

The protein concentration in the eluate was measuredspectrophotometrically at 280 mM:

OD 280 nm in mg LF Adsorbent capacity eluate in eluate mg LF/mladsorbent Purity 4.37 692 35.3 ++++ (approx. 90%)

The adsorbent binds more than 90% of the lactoferrin from the extractmedium after extraction of the precipitated casein in a ratio of 1:100(100 ml extract per ml adsorbent) at a linear flow rate of 15 cm/min.

Example 6 Isolation of Lactoferrin from the Extract Medium AfterExtraction of the Precipitated Casein, by Using Expanded Bed Adsorptionand Different Conductivity Values.

Refining of the extract medium after extraction of the precipitatedcasein comprising lactoferrin (LF) with expanded bed adsorptionchromatography. The binding capacity of the adsorbent is tested atdifferent conductivity values of the extract medium.

An extract medium after extraction of precipitated casein was obtainedfrom a cheese production facility and was analysed to have a pH of 5.4and a conductivity of 120 mS/cm. The extract medium was obtained bywashing the initially obtained cheese curd (precipitated casein) with asolution of sodium chloride whereby a solution of lactoferrin enrichedsalty whey was obtained by draining.

The adsorbent and the column used in this example were the same as thoseused in example 3 above.

Pre-Treatment of the Extract Medium After Extraction of the PrecipitatedCasein

The pH in the salt extract was adjusted to 4.5 with 1N hydrochloricacid. The extract was loaded onto the column un-diluted respectivelydiluted 2, 3 and 4 times with demineralised water.

Loading ratio was 1:50 (980 ml) for the un-diluted salty wheyrespectively 1960, 2940 and 3920 ml for the 2, 3 and 4 times dilutedsalty whey.

The extract medium after extraction of the precipitated casein pH 4.5(salty whey) was loaded onto the column with a linear flow rate of 15cm/min.

Bound impurities were eluted with 50 mN sodium citrate (205, 145, 102and 226 ml respectively).

The lactoferrin was eluted from the adsorbent with 125, 110, 160 and 200ml of 20 mN sodium hydroxide respectively.

The content of protein in each fraction was determined with SDS-PAGE(non-reduced 4-20% tris-glycine gradient gel, Coomassie stained) SeeFIG. 8-11 below.

Results

The protein concentration in the eluate was measuredspectrophotometrically at 280 nm:

Dilution of Conductivity Adsorbent capacity extract medium mS/cm* mgLF/ml adsorbent Purity un-diluted 142 13.6 ++++ (approx. 90%) 1 + 1 7334.5 ++++ (approx. 90%) 1 + 2 54 40.8 ++++ (approx. 90%) 1 + 3 37 45.4++++ (approx. 90%) *Measured after pH-adjustment

Conclusion: The capacity of the adsorbent increases when theconductivity in the extraction media decreases.

Example 7 Isolation of Lactoferrin from the Extract Medium AfterExtraction of the Precipitated Casein, by Using Expanded Bed Adsorptionand Reduced Temperature.

Refining of the extract medium after extraction of the precipitatedcasein comprising lactoferrin (LF) with expanded bed adsorptionchromatography. Adsorption at 15° C.

The extract medium after extraction of the precipitated casein wasobtained from a cheese production facility and was the same as inexample 3.

All conditions except for the temperature at adsorption were the same asdescribed in example 3.

Results

Bound impurities were eluted with 50 mN boric acid pH 9.0 (150 ml).

The lactoferrin was eluted from the adsorbent with 125 ml of 20 mNsodium hydroxide.

The content of protein in each fraction was determined with SDS-PAGE(non-reduced 4-20% tris-glycine gradient gel, Coomassie stained) SeeFIG. 12 below.

The protein concentration in the eluate was measuredspectrophotometrically at 280 nm:

Temp. OD 280 nm mg LF Adsorbent capacity (° C.) in eluate in eluate mgLF/ml adsorbent Purity 15 4.21 474 23.7 ++++ (approx. 90%)

The adsorbent binds practically all the lactoferrin from the extractmedium after extraction of the precipitated casein in a ratio of atleast 1:60 (60 ml extract per ml adsorbent) at 15° C.

1. A process for isolating lactoferrin comprising: i. precipitating acasein precipitate from a product that comprises lactoferrin to a caseinprecipitate, whereby said lactoferrin associates with said caseinprecipitate to form a lactoferrin-casein adsorption product; and ii.extracting said lactoferrin-casein adsorption complex with an extractionmedium, thereby releasing at least part of said lactoferrin from saidlactoferrin-casein adsorption product into the extraction medium.
 2. Theprocess of claim 1, wherein said casein precipitate is obtained frommilk, and wherein said product that comprises lactoferrin is milk, andfurther comprising after step (i) and before step (ii) the step ofwashing said lactoferrin-casein adsorption product with a washingsolution to wash out at least part of undesired entrained whey proteinsfrom the casein precipitate.
 3. The process of claim 2, wherein thewashing solution is drained off the casein precipitate.
 4. The processof claim 1, wherein the lactoferrin containing extraction medium isseparated from the casein precipitate by at least one method selectedfrom the group consisting of draining, centrifugation, filtration andfilter pressing.
 5. The process of claim 1, wherein the extractionmedium provides a change in at least one parameter selected from thegroup consisting of pH, ionic strength, salt content, hydrophobicity,charge, temperature and pressure.
 6. The process of claim 1, wherein theextraction medium is at least one member selected from the groupconsisting of inorganic salts, organic salts, acids, and bases.
 7. Theprocess of claim 1, wherein the extraction medium comprises a final saltconcentration in the range of 0.01-5N.
 8. The process of claim 1,wherein the extraction medium has a pH in the range of 1-5.
 9. Theprocess of claim 1, wherein said product containing lactoferrin isobtained from an animal.
 10. The process of claim 2 wherein said milk isobtained from a transgenic animal producing human or animal lactoferrinin their milk.
 11. The process of claim 1, wherein the animal isselected from the group consisting of herd animals, cows, camels,buffaloes, pigs, horses, deer, sheep, goats, dogs and cats.
 12. Theprocess of claim 1, wherein lactoferrin is isolated by a batch operationor by continuous operation.
 13. The process of claim 4, wherein thelactoferrin in said extraction medium separated from said caseinprecipitate is further isolated by at lease one method selected from thegroup consisting of clarification, concentration and purification. 14.The process of claim 13, wherein said further isolated lactoferrin isclarified by at least one method selected from the group consisting offiltration, membrane filtration, centrifugation and sedimentation. 15.The process of claim 13 or 14, wherein lactoferrin is concentrated by atleast one method selected from the group consisting of membranefiltration, ultrafiltration, nanofiltration, reversed osmosis,evaporation and precipitation.
 16. The process of claim 15, wherein saidconcentrated lactoferring is purified by at least one adsorptiontechnique selected from the group consisting of ion exchange,hydrophobic adsorption, affinity adsorption, mixed mode ligandadsorption, metal chelate adsorption, reversed phase adsorption, gelfiltration and precipitation.
 17. The process of claim 16, wherein theadsorption technique is performed in an expanded bed mode, a packed bedmode or both.
 18. The process of claim 16, wherein said lactoferrin isadsorbed by a resin at a pH in the range of 3-7.
 19. The process ofclaim 16, wherein said lactoferrin is eluted from a resin by increasingpH.
 20. The process of claim 19, wherein the pH for elution of saidlactoferrin adsorbed to a resin is in the range of 8-13.
 21. The processof claim 1, wherein said casein precipitate is obtained from dairyproduct.
 22. The process of claim 1, wherein said lactoferrin isisolated from a casein precipitate obtained from a casein and/orcaseinate production.
 23. The process of claim 1, wherein saidlactoferrin is isolated from a casein precipitate obtained from a cheeseproduction.
 24. A lactoferrin enriched extract comprising lactoferrinisolated according to the process of claim
 1. 25. A compositioncomprising the lactoferrin enriched extract of claim
 24. 26. A methodfor preserving food products comprising combining the lactoferrinenriched extract of claim 24 with a food product.
 27. The method ofclaim 26, further comprising subjecting said combined lactoferrinenriched extract to a preparation step.
 28. The method of claim 26 or27, wherein said food product is a meat product.